The proposed research is a mechanistic study of the action of thrombin. Those areas particularly emphasized are the reaction with the plasma coagulation inhibitors, alpha-2-macroglobulin and antithrombin III, and the clinical anticoagulant, heparin. Reactions with low molecular weight inhibitors will also be investigated as a possible framework for anti-thrombin drug design. The reaction with the physiologic substrate fibrinogen will also be studied. Both kinetic and equilibrium experiments will be performed. Kinetic studies will employ rapid-mix stop-flow and conventional methods and utilize UV-visible spectrophotometry, fluorescence and fluorescence depolarization measurements. The long-range objective is a detailed understanding of the mechanism by which thrombin interacts with its substrates and inhibitors, natural and synthetic.